Krishanu Saha

• PhD
• Professor (Assistant) at University of Wisconsin–Madison

CRISPR-Cas9 gene editing of human cells and tissues holds much promise to advance medicine and biology, but standard editing methods require weeks to months of reagent preparation and selection where much or all of the initial edited samples are destroyed during analysis. ArrayEdit, a simple approach utilizing surface-modified multiwell plates cont...

Statement of significance: CRISPR-Cas9 is a new gene-editing technology for "genome surgery" that is anticipated to treat genetic diseases. This technology uses multiple components of the Cas9 system to cut out disease-causing mutations in the human genome and precisely suture in therapeutic sequences. Biomaterials based delivery strategies could...

Direct reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming is a continuous stochastic process where almost all donor cell...

The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometric...

Proposed rules to protect research subjects will impede progress, say Krishanu Saha and J. Benjamin Hurlbut. Instead, give donors more say in how samples are used.

Chimeric antigen receptor (CAR) T cells have limited efficacy against solid tumors including neuroblastoma. Here, we evaluated whether low-dose radiation delivered by radiopharmaceutical therapy (RPT), known to potentiate immune checkpoint inhibitors, can synergize with CRISPR-edited GD2 TRAC-CAR T cells to improve outcomes in neuroblastoma. We fou...

Glioblastoma (GBM) is an aggressive brain tumor with limited treatment options and poor patient survival, underscoring the need for novel therapeutic strategies and improved preclinical models. Patient-derived tumor spheroids (PDTSs) offer a physiologically relevant in vitro platform for evaluating treatments such as chimeric antigen receptor (CAR)...

After administering genome editors, their efficiency is limited by a multi-step process involving cellular uptake, trafficking, and nuclear import of the vector and its payload. These processes vary widely across cell types and differ depending on the nature and structure of the vector, whether it is a lipid nanoparticle or a different synthetic ma...

CRISPR-Cas9 ribonucleoproteins (RNPs) combined with a nucleic acid template encoding a chimeric antigen receptor (CAR) transgene can edit human cells to produce CAR T cells with precise CAR insertion at a single locus. However, many human cells have adverse innate immune responses to foreign nucleic acids, particularly circular double-stranded DNA...

Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administr...

While chimeric antigen receptor (CAR) T cells have achieved significant success against hematological malignancies, efficacy against neuroblastoma has been limited. Virus-free CRISPR-edited GD2 TRAC-CAR T cells have been developed as a potential means of improving CAR T efficacy but are not curative. Radiopharmaceutical therapy (RPT) is a promising...

Chimeric antigen receptor (CAR) T cell immunotherapy has demonstrated exceptional efficacy against hematological malignancies, but notably less against solid tumors. To overcome this limitation, it is critical to investigate antitumor CAR‐T cell potency in synthetic 3D microenvironments that can simulate the physical barriers presented by solid tum...

Biomedical research has witnessed significant strides in manufacturing chimeric antigen receptor T cell (CAR-T) therapies, marking a transformative era in cellular immunotherapy. Nevertheless, existing manufacturing methods for autologous cell therapies still pose several challenges related to cost, immune cell source, safety risks, and scalability...

Efficient engineering of T cells to express exogenous tumor-targeting receptors such as chimeric antigen receptors (CARs) or T-cell receptors (TCRs) is a key requirement of effective adoptive cell therapy for cancer. Genome editing technologies, such as CRISPR/Cas9, can further alter the functional characteristics of therapeutic T cells through the...

Chimeric antigen receptor (CAR) T cell therapy for solid tumors is challenging not only because of the immunosuppressive tumor microenvironment, but also because of a complex manufacturing process that is difficult to monitor. Manufacturing directly impacts CAR T cell yield, phenotype, and metabolism, which correlate with in vivo potency and persis...

Suspension cell culture and rigid commercial substrates are the most common methods to clinically manufacture therapeutic CAR-T cells ex vivo. However, suspension culture and nano/micro-scale commercial substrates poorly mimic the microenvironment where T cells naturally develop, leading to profound impacts on cell proliferation and phenotype. To o...

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex, with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem c...

Chimeric antigen receptor (CAR) T cell therapy for solid tumors remains challenging due to the complex manufacturing process and the immunosuppressive tumor microenvironment. The manufacturing condition directly impacts CAR T cell yield, phenotype, and metabolism, which correlate with in vivo potency and persistence. Optical metabolic imaging (OMI)...

Natural killer (NK) cells are an appealing off-the-shelf, allogeneic cellular therapy due to their cytotoxic profile. However, their activity against solid tumors remains suboptimal in part due to the upregulation of NK-inhibitory ligands, such as HLA-E, within the tumor microenvironment. Here, we utilize CRISPR-Cas9 to disrupt the KLRC1 gene (enco...

Manufacturing Chimeric Antigen Receptor (CAR) T cell therapies is complex, with limited understanding of how media composition impact T-cell phenotypes. CRISPR/Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant ( TRAC ) gene resulting in TRAC -CAR T cells with an enriched stem...

Spheroids offer a unique opportunity to study personalized disease treatment; however, monitoring of these spheroids relies on time-consuming, end-point analyses. Here we apply 3D QPI using quantitative oblique back illumination microscopy (qOBM) to continuously monitor glioblastoma spheroids treated with radiation, immunotherapies, and chemotherap...

Background and purpose. Chimeric antigen receptor (CAR) T cells have been relatively ineffective against solid tumors. Low-dose radiation which can be delivered to multiple sites of metastases by targeted radionuclide therapy (TRT) can elicit immunostimulatory effects. However, TRT has never been combined with CAR T cells against solid tumors in a...

Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited...

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNC) as a treatment for retinal degeneration. Leber Congenital Amaurosis (LCA16) is a rare pedia...

Introduction: Targeted gene editing is proposed as a therapeutic approach for numerous disorders, including neurological diseases. As the brain is organized into neural networks, it is critical to understand how anatomically connected structures are affected by genome editing. For example, neurons in the substantia nigra pars compacta (SNpc) projec...

Introduction Targeted gene editing is proposed as a therapeutic approach for numerous disorders, including neurological diseases. As the brain is organized into neural networks, it is critical to understand how anatomically connected structures are affected by genome editing. For example, neurons in the substantia nigra pars compacta (SNpc) project...

Background aims: Chimeric antigen receptor (CAR) T cells have demonstrated remarkable efficacy against hematological malignancies; however, they have not experienced the same success against solid tumors such as glioblastoma (GBM). There is a growing need for high-throughput functional screening platforms to measure CAR T-cell potency against soli...

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. I...

Background Natural killer (NK) cells are innate cytotoxic lymphocytes capable of killing virally infected cells and malignant tumors. Unlike T cells, NK cells are human leukocyte antigen (HLA)-agnostic and cause little to no Graft vs. Host Disease in allogenic transfusions, making them excellent candidates for off-the-shelf therapies. However, curr...

Background Chimeric antigen receptor (CAR) T-cells are engineered immune cells that can be taken from a patient and redirected to fight cancer. Recently, we developed a virus-free process to create anti-GD2 CAR T cells using CRISPR-Cas9 ribonucleoproteins that endows cells with a stem cell memory-like and less exhausted phenotype that improves thei...

Introduction: Glioblastoma multiforme (GBM) is the most aggressive and malignant form of brain tumor in adults, with a five-year survival rate of 5%. There are no treatment options known to be significantly effective. CAR T-cell therapy uses patient-derived T-cells that have been engineered to express a chimeric antigen receptor (CAR), which can ta...

Background Chimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; howe...

Genome editing of somatic cells via clustered regularly interspaced short palindromic repeats (CRISPR) offers promise for new therapeutics to treat a variety of genetic disorders, including neurological diseases. However, the dense and complex parenchyma of the brain and the post-mitotic state of neurons make efficient genome editing challenging. I...

Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeted delivery of CRISPR editors. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNC) as a treatment for retinal degeneration. Leber Congenital Amaurosis (LCA16) is a rare pediat...

Point mutations in the KCNJ13 gene cause an autosomal recessive, childhood blindness, Leber congenital amaurosis (LCA16) due to a loss-of-function Kir7.1 channel. In the present study, we investigated the etiology of LCA16 caused by a KCNJ13 missense mutation (c.431T>C, p.Leu144Pro) and explored the activity of two cytosine base editors mRNAs (CBEs...

Photoreceptors are the key functional cell types responsible for the initiation of vision in the retina. Phototransduction involves isomerization and conversion of vitamin A compounds, known as retinoids, and their recycling through the visual cycle. We demonstrate a functional readout of the visual cycle in photoreceptors within stem cell-derived...

The process of reprogramming patient samples to human induced pluripotent stem cells (iPSCs) is stochastic, asynchronous, and inefficient leading to a heterogeneous population of cells. Here, we track the reprogramming status of single patient-derived cells during reprogramming with label-free live-cell imaging of cellular metabolism and nuclear mo...

Expressed emotion (EE), a measure of the family's emotional climate, is a fundamental measure in caregiving research. A core dimension of EE is the level of criticism expressed by the caregiver to the care recipient, with a high level of criticism a marker of significant distress in the household. The Five-Minute Speech Sample (FMSS), the most comm...

Background Chimeric antigen receptor (CAR) T cells traditionally harbor viral vector-based sequences that encode the CAR transgene in the genome. These T cell products have yet to show consistent anti-tumor activity in patients with solid tumors. Further, viral vector manufacturing is resource intensive, suffers from batch-to-batch variability, and...

Glioblastoma (GBM) is an aggressive form of brain cancer that has no effective treatments and a prognosis of only 12-18 months. Immune effector T cells are a promising therapy due to their innate cytotoxicity. In addition, engineering chimeric antigen receptors (CAR) to target tumor-associated or neo-antigens can lend high specificity. The ability...

Biomedicine is being revolutionized by many CRISPR/Cas systems that introduce programmable edits to nearly any gene in the human genome. Nuclease-based CRISPR/Cas editors can produce on-target genomic changes but can also generate unwanted genotoxicity and adverse events, in part by cleaving non-targeted sites in the genome. Additional translationa...

Glioblastoma (GBM) is an aggressive brain cancer without effective treatments. CAR-T cells targeted to tumor-associated antigens offer promise for treating GBM. Here, we used cellular impedance assays to compare the cytolytic potency and kinetics of conventional viral vs non-viral CRISPR engineered GD2 CAR-T cells against glioma stem cells (GSC), a...

The recent discovery and subsequent development of the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) platform as a precise genome editing tool have transformed biomedicine. As these CRISPR-based tools have matured, multiple stages of the gene editing process and the bioengineering of human cells and tissu...

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tiss...

There is a critical need for adjuvants that can safely elicit potent and durable T cell-based immunity to intracellular pathogens. Here, we report that parenteral vaccination with a carbomer-based adjuvant, Adjuplex (ADJ), stimulated robust CD8 T-cell responses to subunit antigens and afforded effective immunity against respiratory challenge with a...

The function of a T cell depends on its subtype and activation state. Here, we show that imaging of the autofluorescence lifetime signals of quiescent and activated T cells can be used to classify the cells. T cells isolated from human peripheral blood and activated in culture using tetrameric antibodies against the surface ligands CD2, CD3 and CD2...

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct...

Gene correction of multiple alleles for compound heterozygous recessive or polygenic diseases is a promising therapeutic strategy. However, the targeting of multiple alleles using genome editors in a single cell could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Spy Cas9-based S1mplex...

Dominantly inherited disorders are not typically considered to be therapeutic candidates for gene augmentation. Here, we utilized induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations in BEST1. G...

Purpose : CRISPR-Cas9 gene-editing technology efficiently targets specific loci within the human genome using sequence-specific guide RNAs. The present study utilized the gene-editing strategy to treat Leber congenital amaurosis (LCA16) caused by the mutations in the KCNJ13 gene that encodes for the inwardly rectifying potassium channel (Kir7.1). O...

This essay introduces a collection of articles gathered under the theme of “law, science, and constitutions of life.” Together, they explore how revolutions in notions of what biological life is are eliciting correspondingly revolutionary imaginations of how life should be governed. The central theoretical contribution of the collection is to furth...

Efficient delivery of hydrophilic drugs, nucleic acids, proteins, and any combination thereof is essential for various biomedical applications. Herein, we report a straightforward, yet versatile approach to efficiently encapsulate and deliver various hydrophilic payloads using a pH-responsive silica–metal–organic framework hybrid nanoparticle (SMOF...

Dominantly inherited disorders are not typically considered therapeutic candidates for gene augmentation. Here, we utilized patient-specific induced pluripotent stem cell-derived retinal pigment epithelium (iPSC-RPE) to test the potential of gene augmentation to treat Best disease, a dominant macular dystrophy caused by over 200 missense mutations...

Delivery technologies for the CRISPR-Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing system often require viral vectors, which pose safety concerns for therapeutic genome editing¹. Alternatively, cationic liposomal components or polymers can be used to encapsulate multiple CRISPR components into large particles...

An international regulatory commission convened by scientific academies is a premature and problematic approach to governing human germline genome editing. Given the complex, international landscape of genome editing and significant cross-national differences among regulatory cultures, deferring to a single commission to set the agenda for global g...

Reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) generates valuable resources for disease modeling, toxicology, cell therapy, and regenerative medicine. However, the reprogramming process can be stochastic and inefficient, creating many partially reprogrammed intermediates and non-reprogrammed cells in addition to full...

The impact of the FMR1 premutation on human health is the subject of considerable controversy. A fundamental unanswered question is whether carrying the premutation allele is directly correlated with clinical phenotypes. A challenging problem in past genotype-phenotype studies of the FMR1 premutation is ascertainment bias, which could lead to inval...

Gene therapy holds great promise for the treatment of many diseases, but clinical translation of gene therapies has been slowed down by the lack of safe and efficient gene delivery systems. Here, we report two versatile redox-responsive polyplexes (i.e., cross-linked and non-crosslinked) capable of efficiently delivering a variety of negatively cha...

Recent advancements in immunooncology have resulted in the generation of novel therapies such as chimeric antigen receptor (CAR) T cells, which have revolutionized the treatment of pediatric patients with relapsed or refractory B-cell acute lymphoblastic leukemia. The journey of tisagenlecleucel (formerly CTL019) from early preclinical success to t...

Professor Beriain's criticism rests on a narrow conception of human dignity pertaining only to individuals within a society. The social relations and norms that underpin human dignity are treated as mere group interests that are secondary to the dignity of the individual. In our view, this is a false dichotomy.

New gene editing tools like CRISPR-Cas9 enable precision genome engineering within cell lines, primary cells, and model organisms, with some formulations now entering the clinic. "Precision" applies to various aspects of gene editing, and can be tailored for each application. Here we review recent advances in four types of precision in gene editing...

A new infrastructure is urgently needed at the global level to facilitate exchange on key issues concerning genome editing. We advocate the establishment of a global observatory to serve as a center for international, interdisciplinary, and cosmopolitan reflection. This article is the first of a two-part series.

A new infrastructure is urgently needed at the global level to facilitate exchange on key issues concerning genome editing. We advocate the establishment of a global observatory to serve as a center for international, interdisciplinary, and cosmopolitan reflection. This article is the second of a two-part series.

T cells can have different activities based on receptor expression and cytokine production. Current methods to classify and assess immune cell behavior include flow cytometry and immunohistochemistry, which require immune cell labeling and tissue fixation. A non-invasive method for determining T cell behavior is needed to study immune cell behavior...

The gradual accumulation of amyloid-β (Aβ) is a neuropathologic hallmark of Alzheimer's disease (AD); playing a key role in disease progression. Aβ is generated by the sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases, with BACE-1 (β-site APP cleaving enzyme-1) cleavage as the rate limiting step. CRISPR/Cas9 guided gene-...

Many avenues exist for human pluripotent stem cells (hPSCs) to impact medical care, but they may have their greatest impact on the development of precision medicine. Recent advances in genome editing and stem cell technology have enabled construction of clinically-relevant, genotype-specific "disease-in-a-dish" models. In this review, we outline th...

Genome-edited human pluripotent stem cells (hPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. We present and characterize a robust method for rapid, scarless introduction or correction of disease-associated variants in hPSCs using CRISPR/Cas9. Utilizing non-integrated plasmid vectors that express a purom...

Writing specific DNA sequences into the human genome is challenging with non-viral gene-editing reagents, since most of the edited sequences contain various imprecise insertions or deletions. We developed a modular RNA aptamer-streptavidin strategy, termed S1mplex, to complex CRISPR-Cas9 ribonucleoproteins with a nucleic acid donor template, as wel...

The next generation of therapeutic products to be approved for the clinic is anticipated to be cell therapies, termed "living drugs" for their capacity to dynamically and temporally respond to changes during their production ex vivo and after their administration in vivo. Genetically engineered chimeric antigen receptor (CAR) T cells have rapidly d...

Emerging manufacturing processes to generate regenerative advanced therapies can involve extensive genomic and/or epigenomic manipulation of autologous or allogeneic cells. These cell engineering processes need to be carefully controlled and standardized to maximize safety and efficacy in clinical trials. Engineered biomaterials with smart and tuna...

Millions of people globally are at high risk for neurodegenerative disorders, infertility or having children with a disability as a result of the Fragile X (FX) premutation, a genetic abnormality in FMR1 that is underdiagnosed. Despite the high prevalence of the FX premutation and its effect on public health and family planning, most FX premutation...

Reprogramming of human somatic cells to induce pluripotent stem cells (iPSCs) generates valuable precursors for disease modeling and regenerative medicine. However, the reprogramming process can be inefficient and noisy, creating many partially reprogrammed cells in addition to fully reprogrammed iPSCs. To address these shortcomings, we developed a...

Genome editing in human pluripotent stem cells (hPSCs) enables the generation of reporter lines and knockout cell lines. Zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9 technology have recently increased the efficiency of proper gene editing by creating double strand breaks (DSB) at defined sequences...

Human patient-derived induced pluripotent stem cells (hiPSCs) provide unique opportunities for disease modeling and drug development. However, adapting hiPSCs or their differentiated progenies to high throughput assays for phenotyping or drug screening has been challenging. Fragile X syndrome (FXS) is the most common inherited cause of intellectual...

Background: Non-viral gene-editing of human cells using the CRISPR-Cas9 system requires optimized delivery of multiple components. Both the Cas9 endonuclease and a single guide RNA, that defines the genomic target, need to be present and co-localized within the nucleus for efficient gene-editing to occur. This work describes a new high-throughput s...